Coccidiocidal combinations

ABSTRACT

Synergistic coccidiocidal combinations comprise two ionophorous fermentation-derived coccidiocides, each of which is administered to poultry in a concentration too low to produce satisfactory coccidiocidal effect alone. Preferred combinations include monensin and A204, monensin and lasalocid, and monensin and A28086.

BACKGROUND OF THE INVENTION

This invention belongs to the field of poultry husbandry and providesnew methods and compositions for the control of coccidiosis of poultry.The damage done by coccidiosis is well known. All of the economicallyreared poultry species are subject to infections of coccidia, whichcause severe losses unless controlled. Coccidial infections not onlycause the infected birds to gain weight more slowly than normal, and toconvert their feed inefficiently, but often result in the deaths oflarge numbers of birds.

Like most parasitic infections, coccidiosis is most frequent and mostdamaging in flocks of birds reared in conditions of high populationdensity. Since these conditions are also the most economical, modernefficient poultry operations are very likely to suffer severely fromcoccidiosis.

All of the coccidiocides used in this invention are known in the animalhealth art. References describing the compounds and their preparationwill be cited below. The novelty of the present invention resides in thediscovery that the compounds, when combined in proper amounts orconcentrations, are synergistic. The coccidiocidal effect of thecombinations is greater than the effect which could be expected from theknown effects of the components of the combination, when used alone.Thus, use of the combinations allows the poultry grower to obtaineconomically satisfactory coccidiocidal effect with the administrationof unusually small amounts of the coccidiocides. Small dosages not onlyhave the obvious economic benefit, but also are likely to result inreduced residues of coccidiocide in the edible tissues of the poultry.

SUMMARY OF THE INVENTION

This invention provides a coccidiocidal composition for poultrycomprising a coccidiocidally-ineffective concentration of each of twoionophorous fermentation-derived coccidiocides, which coccidiocides aresynergistically coccidiocidally effective in combination, together witha poultry feedstuff or drinking water.

The preferred ionophorous fermentation-derived coccidiocides are asfollows, together with the preferred concentrations in which they are tobe used.

From about 20 to about 70 parts per million by weight (ppm) of monensin

From about 4 to about 20 ppm of A204

From about 80 to about 120 ppm of nigericin

From about 30 to about 50 ppm of A28086

From about 10 to about 20 ppm of A28695

From about 25 to about 50 ppm of lasalocid

The terms monensin, A204, nigericin, A28086, A28695 and lasalocid areused herein to include the alkali metal, alkaline earth metal, ammoniumand primary, secondary and tertiary C₁ -C₄ alkylamine and C₂ -C₄hydroxyalkylamine salts thereof.

Preferred compositions include those wherein one of the coccidiocides ismonensin, and its concentration is from about 20 to about 70 ppm.

Specific preferred compositions which are desirable embodiments of theinvention include those wherein the following coccidiocides are used inthe following concentrations in feed or drinking water:

From about 20 to about 70 ppm of monensin and from about 4 to about 20ppm of A204.

From about 20 to about 70 ppm of monensin and from about 80 to about 120ppm of nigericin.

From about 20 to about 70 ppm of monensin and from about 30 to about 50ppm of A28086.

From about 20 to about 70 ppm of monensin and from about 10 to about 20ppm of A28695.

From about 20 to about 70 ppm of monensin and from about 25 to about 50ppm of lasalocid.

From about 30 to about 50 ppm of A28086 and from about 10 to about 20ppm of A28695.

From about 4 to about 20 ppm of A204 and from about 10 to about 20 ppmof A28695.

Particularly preferred specific compositions include those wherein thefollowing coccidiocides are used in the following concentrations:

From about 25 to about 50 ppm of monensin and from about 5 l to about16.5 ppm of A204.

From about 25 to about 50 ppm of monensin and about 100 ppm ofnigericin.

From about 25 to about 50 ppm of monensin and from about 40 to about 45ppm of A28086.

From about 25 to about 50 ppm of monensin and about 15 ppm of A28695.

From about 25 to about 50 ppm of monensin and about 37 ppm of lasalocid.

From about 40 to about 45 l ppm of A28086 and about 15 ppm of A28695.

From about 5 to about 16.5 ppm of A204 and about 15 ppm of A28695.

This invention also provides a coccidiocidal method for poultry whichcomprises orally administering to the poultry a composition as describedabove.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Most of the coccidiocides discussed here comprise two or more factors,like most products of fermentation origin. The various factors are allusable in this invention. As is usual in the art, the names of thecoccidiocides are used herein to refer to the unresolved mixture offactors produced by fermentation, when a specific factor is notindicated.

The coccidiocides used in the present invention have been disclosed inthe prior art. The compounds will be reviewed and references where theirdescriptions and preparations may be found will be named.

Monensin is described in U.S. Pat. No. 3,501,568, where it is calledA3823 complex.

Another patent, U.S. Pat. No. 3,832,358, shows a modified form ofmonensin known as deshydroxymethylmonensin, which is likewise functionalin the present invention.

A204 is disclosed by U.S. Pat. No. 3,705,238.

A28695 is described in U.S. Pat. No. 3,839,558.

Lasalocid, formerly known as X-537A, is discussed in U.S. Pat. No.3,719,753. Lasalocid derivatives are disclosed and described in U.S.Pat. Nos. 3,873,715, 3,836,516, and 3,715,372. All of the derivatives oflasalocid, of course, may be used in this method equally as effectivelyas the parent compound.

Nigericin is described in U.S. Pat. No. 3,794,732.

A28086 is described in U.S. Patent Applications No. 569,740 and 569,719,both filed Apr. 21, 1975. The disclosures of both applications areherein incorporated by reference.

The following matter describes A28086 and the microorganisms andfermentation processes which produce it.

Antibiotic A-28086 factor A crystallizes from acetone-water, and meltsat about 98°-100° C., resolidifies and remelts at about 195°-200° C.Elemental analysis of factor A gave the following average percentagecomposition: carbon, 66.69 percent; hydrogen, 9.85 percent; oxygen,23.10 percent. The empirical formula proposed for factor A is C₄₃ H₇₂O₁₁.

Factor A has an apparent molecular weight of 764 as determined by massspectrometry.

The infrared spectrum of factor A in chloroform showed the followingabsorption maxima: 2.85, 3.34, 5.83, 6.82, 7.22, 7.53 (weak), 7.78(weak), 8.75 (strong), 8.95 (strong), 9.15, 9.50 (strong), 9.55(strong), 9.60, 9.85, 10.15, 10.45, and 10.70 (weak) microns.

The ultraviolet spectrum of factor A in ethanol shows only endabsorption below 220 mμ.

The nuclear magnetic resonance spectrum of A-28086 factor A indeuterochloroform showed the following characteristics: δ 6.01, 4.21,4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3.57, 3.55, 2.83, 2.76, 2.74, 2.68,2.66, 2.58, 2.56, 2.30, 2.22, 2.17, 2.10, 2.05, 1.96, 1.90, 1.85, 1.70,1.62, 1.60, 1.47, 1.39, 1.31, 1.25, 1.18, 0.95, 0.93, 0.90, 0.88, 0.85,0.77, 0.75, 0.73, 0.68, and 0.66 ppm.

Antibiotic A-28086 factor A, crystallized from acetone-water, has thefollowing characteristic X-ray powder diffraction pattern (Cu⁺⁺radiation, 1.5405λ, nickel filter, d = interplanar spacing inangstroms):

    ______________________________________                                                      Relative                                                               d      Intensity                                                       ______________________________________                                               12.00  100                                                                    10.10  50                                                                     9.25   90                                                                     8.00   40                                                                     7.50   15                                                                     6.92   90                                                                     6.40   40                                                                     5.98   05                                                                     5.68   15                                                                     5.20   40                                                                     4.98   40                                                                     4.62   40                                                                     4.21   20                                                                     3.48   10                                                              ______________________________________                                    

The specific rotation of antibiotic A-28086 factor A is -54° (c=0.2,methanol), when determined at a temperature of 25° C. This specificrotation is an average value, based on several determinations.

Electrometric titration of factor A in 80% aqueous dimethylformamideindicated the presence of a titratable group with a pK_(a) value of 7.9.

Antibiotic A-28086 factor A is soluble in a variety of organic solventssuch as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethylacetate, chloroform, acetone, and benzene; but is only slightly solublein non-polar organic solvents such as hexane; and is insoluble in water.

Antibiotic A-28086 factor B is a white crystalline compound (fromacetone-water) which has a melting point of about 150°-153° C.

As determined by high-resolution mass spectrometry, factor B has anapparent molecular weight of 762 and a proposed empirical formula of C₄₃H₇₀ O₁₁.

The infrared spectrum of factor B in chloroform showed the followingabsorption maxima: 2.82, 3.30, 5.77, 5.85, 6.80, 7.20, 7.50 (weak), 7.72(weak), 7.80 (weak), 8.57 (strong), 8.68, 8.90 (strong), 9.10, 9.50,9.83 (strong), 9.90, 10.10, 10.17 (strong), 10.43 (weak), 10.80 (weak),11.20 (weak), 11.35 (weak), 11.73 (weak), and 12.03 (weak) microns.

The ultraviolet spectrum of factor B in ethanol shows an absorptionmaximum at 220 mμ (E_(1cm) ^(1%) 32 137.5; ε=10,477).

The nuclear magnetic resonance spectrum of A-28086 factor B indeuterochloroform showed the following characteristics: δ 7.20, 7.09,6.26, 6.15, 4.19, 4.12, 4.05, 3.95, 3.89, 3.78, 3.62, 3.59, 3.52, 3.48,2.81, 2.73, 2.63, 2.54, 2.52, 1.99, 1.91, 1.84, 1.71, 1.67, 1.64, 1.55,1.43, 1.33, 1.18, 1.11, 0.96, 0.94, 0.90, 0.87, 0.84, 0.77, 0.74, and0.68 ppm.

Antibiotic A-28086 factor B is soluble in a variety of organic solventssuch as, for example, methanol, ethanol, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene; but is onlyslightly soluble in nonpolar organic solvents such as hexane; and isinsoluble in water.

Although the chemical structure of A-28086 antibiotic factor B has notbeen elucidated, the physical-chemical data thus far available indicatethat factor B has a single carboxylic acid moiety, two ketone moieties,and one or more hydroxyl moieties.

Antibiotic A-28086 factor D is a white crystalline material (fromwater-acetone) with a melting point of about 96°-98° C. A-28086 factor Dhas an apparent molecular weight of 778, as determined byhigh-resolution mass spectrometry.

The elemental composition of the peak in the mass spectrum of the sodiumsalt of A-28086 factor D was observed to be 800.5050 (Calcd for C₄₄ H₇₃O₁₁ Na = 800.5050). In the mass spectrum of A-28086 factor D free acid,a small peak at 778 and a larger peak at 760.5117 (Calcd for C₄₄ H₇₂ O₁₀= 760.5125) were observed. The m/e 760 in the mass spectrum of the freeacid results from the loss of water from the molecular ion. Themolecular-ion composition of A-28086 factor D free acid is, therefore,C₄₄ H₇₄ ₁₁.

The empirical formula proposed for A-28086 factor D is C₄₄ H₇₄ O₁₁.Elemental analysis of factor D gave the following percentagecomposition: carbon, 67.59 percent; hydrogen 9.38 percent; oxygen, 22.77percent.

The theoretical percentage composition for C₄₄ H₇₄ O₁₁ is: carbon, 67.87percent; hydrogen, 9.51 percent; oxygen, 22.77 percent.

The infrared absorption spectrum of A-28086 factor D contains thefollowing observable absorption maxima: 2.89, 3.39, 3.43, 3.50, 5.88,6.90, 7.27, 7.60, 7.84, 9.00, 9.26, 9.62, 10.31, 10.58, 11.10, and 11.49microns.

A-28086 factor D in 95 percent aqueous ethanol shows no ultravioletabsorption.

The nuclear magnetic resonance spectrum of A-28086 factor D indeuterochloroform showed the following characteristics: δ 6.00, 4.20,4.10, 4.00, 3.98, 3.92, 3.86, 3.83, 3.79, 3.67, 3.64, 3.57, 3.54, 2.88,2.81, 2.71, 2.62, 2.58, 2.48, 2.43, 2.37, 2.29, 2.21, 2.15, 2.10, 2.04,1.97, 1.89, 1.83, 1.76, 1.68, 1.61, 1.58, 1.55, 1.47, 1.39, 1.30, 1.25,1.18, 0.95, 0.90, 0.88, 0.84, 0.74, and 0.68 ppm.

Antibiotic A-28086 factor D, crystallized from acetone-water, has thefollowing characteristic X-ray powder-diffraction pattern (Cu₊₊radiation, 1.5405λ, nickel filter, d = interplanar spacing inangstroms):

    ______________________________________                                                      Relative                                                               d      Intensity                                                       ______________________________________                                               12.40  100                                                                    10.20  70                                                                     8.85   90                                                                     7.80   30                                                                     6.80   10                                                                     6.30   100                                                                    5.70   20                                                                     5.35   20                                                                     5.10   20                                                                     4.90   10                                                                     4.65   20                                                                     4.45   40                                                                     4.20   30                                                                     3.30   10                                                                     3.15   10                                                                     2.99   05                                                                     2.77   05                                                                     2.28   05                                                              ______________________________________                                    

The specific rotation of antibiotic A-28086 factor D is -56°(c = 0.1,methanol), when determined at a temperature of 25° C.

Electrometric titration of A-28086 factor D in 80 percent aqueousdimethylformamide indicated the presence of a titratable group with apK_(a) value of 8.67.

Antibiotic A-28086 factor D is soluble in a variety of organic solventssuch as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethylacetate, chloroform, acetone and benzene. A-28086 factor D is onlyslightly soluble in nonpolar organic solvents such as hexane and isinsoluble in water.

Antibiotic A-28086 factor D has an acid function capable of formingsalts and ester derivatives and at least one hydroxyl group capable ofesterification.

The R_(f) values of antibiotic A-28086 factors A, B and D in variouspaper-chromatographic systems, using Bacillus subtilis ATCC 6633 as adetection organism, are given in Table I.

                  TABLE I                                                         ______________________________________                                        R.sub.f Value                                                                 Factor A                                                                             Factor B  Factor D  Solvent System                                     ______________________________________                                        0.11   0.09      0.10      Water saturated with                                                          methyl isobutyl                                                               ketone (MIBK)                                      0.41   0.16      0.26      Water saturated with                                                          MIBK plus 2% p-tolu-                                                          enesulfonic acid and                                                          1% piperidine                                      0.54   0.46      0.36      Water:methanol:ace-                                                           tone (12:3:1)-                                                                adjusted to pH 10.5                                                           with NH.sub.4 OH and then                                                     lowered to pH 7.5                                                             with H.sub.3 PO.sub.4                              0.48   0.36      0.29      1% MIBK, 0.5% NH.sub.4 OH                                                     in water                                           0.15   0.33      0.25      17.4 g K.sub.2 HPO.sub.4, 30 ml                                               ethanol per liter of                                                          water                                              0.24   0.51      0.26      Benzene saturated                                                             with water                                         0.24   0.11      0.09      Water                                              0.75   0.61      0.64      Water:MIBK:ethyl                                                              acetate (98:1:1)                                   ______________________________________                                    

In Table II are given the R_(f) values for antibiotic A-28086 factors A,B and D in two thin-layer-chromatographic systems on silica gel(precoated plates, E. Merck, Darmstadt, F-254, layer thickness 0.25 mm),again using B. subtilis ATCC 6633 as a detection organism.

                  TABLE II                                                        ______________________________________                                        R.sub.f Values                                                                Factor A                                                                              Factor B   Factor D   Solvent System                                  ______________________________________                                        0.24    0.42       0.26       Benzene-ethyl ace-                                                            tate (3:2)                                      0.54    0.34       0.66       Ethyl acetate-di-                                                             ethylamine (95:5)                               ______________________________________                                    

Another substance, arbitrarily designated as A-28086-I, is co-producedwith the antibiotic A-28086 complex. Although A-28086-I is notmicrobiologically active, it is structurally related to the A-28086antibiotic factors. A-28086-I is a white crystalline compound (fromacetone-water) and has a melting point of about 160°-162° C. Comparativestudies of the NMR spectra and other properties of A-28086-I andsynthetically-prepared A-28086 factor A methyl ester give evidence thatA-28086-I is the methyl ester, formed on the acid group, of A-28086factor A or a closely related compound such as a stereoisomer.

Although A-28086-I initially co-precipitates with the active A-28086antibiotic factors, it is readily separated from them by silica gelchromatography. A-28086-I has an approximate R_(f) value of 0.53 onsilica gel thin-layer chromatography with ethyl acetate as the elutingsolvent and using vanillin spray reagent (3% vanillin in methanol + 0.5ml conc H₂ SO₄ per 100 ml of solution) for detection. After sprayingwith vanillin and heating, A-28086-I gives a blue spot while the A-28086antibiotic factors give bright pink spots which quickly turn darkbrownish-blue.

The compounds are produced by culturing an A-28086-producing strain ofSteptomyces aureofaciens, either NRRL 5758 or NRRL 8092, under submergedaerobic conditions in a suitable culture medium until substantialantibiotic activity is produced. The antibiotics are recovered byemploying various isolation and purification procedures commonly usedand understood in the art.

The organisms are classified as strains of S. aureofaciens Duggar, asdescribed by E. B. Shirling and D. Gottlieb in "Cooperative Descriptionof Type Cultures of Streptomyces. III. Additional Species Descriptionsfrom First and Second Studies," Intern. Bull. Systematic Bacteriol. 18,279-392 (1968).

CHARACTERIZATION OF A-28086-PRODUCING STRAIN NRRL 5758 Morphology

Sporulating aerial hyphae consist of hooks, loops and open spirals. Alsoobserved was morphology representative of the rectus flexibilis type.Spores are short and cylindrical and are in chains of 10-50 spores. Thespores measure 1.3μ × 1.75μ with a range of 1.3μ to 1.95 × 1.3μ.

The spore surface, as observed by electron microscopy, is smooth.

    ______________________________________                                        Cultural Characteristics of NRRL                                              5758 on Various Media                                                         Medium          Characteristics                                               ______________________________________                                        ISP No. 2 (yeast ex-                                                                          Growth-abundant; reverse mod-                                 tract-malt extract)                                                                           erate yellow [11K3]; fair-to-                                                 good aerial mycelium and                                                      sporulation; white (W) 13 ba                                                  and dark gray (Gy) 3 ih; no                                                   soluble pigment.                                              ISP No. 3 (oatmeal)                                                                           Growth-good; reverse grayish                                                  yellow [11B2]; fair aerial                                                    mycelium (Gy) dark gray,                                                      3 ih; slight brown soluble                                                    pigment.                                                      ISP No. 4 (inorganic                                                                          Growth-abundant; reverse                                      salts-starch agar)                                                                            light yellow brown [12E5];                                                    aerial mycelium and spore                                                     (W) purplish white 13 ba                                                      to (Gy) light gray d; no                                                      soluble pigment.                                              ISP No. 5 (glycerol-                                                                          Growth-good; reverse pale                                     asparagine agar)                                                                              yellow-green [10B1]; good                                                     aerial mycelium and spores                                                    (Gy) yellowish gray 2 dc,                                                     to light grayish reddish                                                      brown 5 fe; no soluble                                                        pigment.                                                      Tomato paste-oatmeal                                                                          Growth-abundant; reverse                                      agar            light yellow brown [13H7];                                                    fair to good aerial mycelium                                                  and spores (W) white a to                                                     (Gy) medium gray g; very                                                      slight brown soluble pigment.                                 Glycerol-glycine agar                                                                         Growth-abundant; reverse                                                      dark grayish yellow [12I6];                                                   good aerial mycelium and                                                      spores (Y) pale yellow                                                        2 db; no soluble pigment.                                     Glucose-asparagine                                                                            Growth-abundant; reverse                                      agar            grayish greenish yellow                                                       [12E2]; abundant aerial                                                       mycelium and spores (Gy)                                                      yellowish gray 2 dc; very                                                     slight brown soluble                                                          pigment.                                                      Nutrient agar   Growth-good; reverse gray-                                                    ish yellow [12B2]; aerial                                                     myceliuman spores, no                                                         color assignment because of                                                   poor growth; no soluble                                                       pigment.                                                      Bennett's agar  Growth-abundant, reverse                                                      grayish yellow [12K3];                                                        scant aerial mycelium and                                                     spores (Gy) yellowish gray                                                    2 dc; no soluble pigment.                                     Calcium malate  Growth-good; reverse gray-                                    agar            ish brown [15C8]; no aerial                                                   mycelium or sporulation;                                                      brown soluble pigment.                                                        Area by inoculum cleared.                                     Czapek's solution                                                                             Growth-poor; no color                                         agar            assignment due to poor                                                        growth.                                                       Emerson's agar  Growth-abundant; reverse                                                      grayish yellow [11J5]; no                                                     aerial mycelium or spores;                                                    no soluble pigment.                                           Tyrosine agar   Growth-abundant; reverse                                                      light olive brown [14C4];                                                     abundant aerial mycelium                                                      and spores from (W) b                                                         (center) to (Gy) light                                                        brownish gray 3 fe (margin);                                                  very slight brown soluble                                                     pigment.                                                      Tryptone-yeast agar                                                                           Growth-scant; no-color                                                        assignment.                                                   ______________________________________                                    

The NRRL 5758 organism was studied for selected physiological propertiesin accordance with standard procedures. The properties observed andcharacteristics found were as follows:

    ______________________________________                                        Property Observed Characteristic                                              ______________________________________                                        Action on milk    Milk peptonized, white                                                        growth ring; cleared area                                                     tannish yellow-pH reaction                                                    5.7                                                         Nitrate reduction Positive                                                    Nutrient gelatin  30% liquefaction at 14                                                        days                                                        Melanin pigment                                                               production on:                                                                Tyrosine-agar     Very weak positive (pig-                                    slants            ment after 4 days)                                          Difco peptone     Negative                                                    yeast extract                                                                 Iron agar slants                                                              Tryptone-yeast-   Negative                                                    extract broth                                                                 Temperature require-                                                                            26-30° C-good growth; 30-                            ments (ISP medium 37° C.-excellent growth                              No. 2 yeast extract                                                                             and sporulation; 45° C.-                             malt extract slants)                                                                            slight vegetative growth;                                                     reddish soluble pigment.                                    ______________________________________                                    

The results of carbon utilization tests carried out with organism NRRL5758 are set forth below. The symbols used to indicate growth responseare:

    ______________________________________                                        +         good growth, positive utilization                                   (+)       poor to fair growth                                                 (-)       faint growth, probably no utilization                               -         no growth, no utilization                                           Carbon Source   Response                                                      ______________________________________                                        D-glucose       +                                                             L-arabinose     +                                                             D-xylose        +                                                             D-fructose      +                                                             sucrose         -                                                             D-mannitol      -                                                             i-inositol      +                                                             rhamnose        +                                                             raffinose       -                                                             C control                                                                     (no carbohydrate)                                                                             -                                                             ______________________________________                                    

A second new A-28086-producing organism was derived from S. aureofaciensNRRL 5758 by a series of natural selections, followed by chemicalmutation, and is identified as NRRL 8092.

CHARACTERIZATION OF A-28086-PRODUCING STRAIN NRRL 8092 Morphology

On medium ISP No. 7 (tyrosine agar) the culture produces occasionalhooks, but mainly produces short, straight sporophores. Spore chains areless than 10 spores per chain, usually 4-7 spores per chain. Shortstraight spore chains were observed in the following media: ISP No. 3,Czapek's-solution agar and ISP No. 5. Abundant coremia were observed onEmerson's agar. Electron microscope observations were made on tyrosineagar (ISP No. 7) and glucose-asparagine agar. Spores are smooth andrange in size from 1.2 to 2.0μ in length and about 1.0μ in diameter. Theaverage spore size is 1.6μ × 1.0μ.

    ______________________________________                                        Cultural Characteristics of NRRL 8092                                         on Various Media                                                              Medium          Characteristics                                               ______________________________________                                        ISP No. 2 (yeast ex-                                                                          Growth-fair; reverse light                                    tract-malt extract)                                                                           yellow brown [12H8]; fair                                                     aerial mycelium; poor                                                         sporulation; aerial pale                                                      gray [11A1]; no soluble                                                       pigment.                                                      ISP No. 3 (oatmeal)                                                                           Growth-sparse; reverse hya-                                                   line; no aerial mycelium;                                                     no soluble pigment.                                           ISP No. 4 (inorganic                                                                          Growth-moderate; reverse                                      salts-starch agar)                                                                            grayish yellow [11B2]; scant                                                  aerial mycelium and sporula-                                                  tion; aerial pale yellow                                                      gray [10A1]; no soluble                                                       pigment.                                                      ISP No. 5 (glycerol-                                                                          Growth-moderate; reverse                                      asparagine agar)                                                                              pale yellow [10F2]; fair                                                      aerial mycelium; scant sporu-                                                 lation; aerial white [10A1];                                                  no soluble pigment.                                           Tomato paste-oatmeal                                                                          Growth moderate; reverse gray-                                agar            ish green-yellow; aerial                                                      mycelium fair; moderate                                                       sporulaton, light pale gray                                                   [53A2]; no soluble pigment.                                   Glycerol-glycine agar                                                                         Growth-abundant; reverse                                                      grayish yellow [11E4]; mod-                                                   erate aerial mycelium, white                                                  [10A1]; no sporulation; no                                                    soluble pigment.                                              Glucose-asparagine agar                                                                       Growth-moderate; reverse pale                                                 yellow ( 10F2]; moderate                                                      aerial mycelium and sporu-                                                    lation, white [10A1]; no                                                      soluble pigment.                                              Nutrient agar   Growth-sparse; reverse pale                                                   yellow [10B2]; no aerial                                                      mycelium; no soluble pigment.                                 Bennett's agar  Growth-fair; reverse medium                                                   yellow pink [11A7]; very                                                      scant aerial mycelium; no                                                     sporulation; no soluble pig-                                                  ment.                                                         Calcium malate agar                                                                           Growth-very scant, hyaline;                                                   no aerial mycelium; no solu-                                                  ble pigment.                                                  Czapek's solution agar                                                                        Growth-very scant; reverse                                                    hyaline; no aerial mycelium;                                                  no soluble pigment.                                           Emerson's agar  Growth-moderate; reverse                                                      grayish yellow [11I5]; spotty                                                 aerial mycelium; no sporula-                                                  tion; no soluble pigment.                                     Tyrosine agar   Growth-moderate; reverse                                                      light yellow brown [12H6];                                                    moderate aerial mycelium,                                                     light pale gray margin                                                        [53A2], center near white,                                                    and moderate sporulation; no                                                  soluble pigment.                                              Tryptone-yeast agar                                                                           Growth-very scant, hyaline;                                                   no aerial mycelium; no soluble                                                pigment.                                                      ______________________________________                                    

Organism NRRL 8092 was also studied for selected physiologicalproperties in accordance with standard procedures. The propertiesobserved and characteristics found were as follows:

    ______________________________________                                        Property Observed                                                                            Characteristic                                                 ______________________________________                                        Action on milk Peptonized (90%); pale-                                                       yellow growth ring, cleared                                                   area pale yellow--pH reaction                                                 4.6                                                            Nitrate reduction                                                                            Positive                                                       Nutrient gelatin                                                                             50% hydrolyzed at 14 days                                      Melanin pigment                                                               production on:                                                                Tyrosine-agar  Very weakly positive                                           slants                                                                        Tryptone-yeast-                                                                              Negative                                                       extract broth                                                                 Carrot plug    Abundant growth, pale                                                         yellow; no aerial mycelium                                     Potato plug    Abundant growth, grayish                                                      white; no aerial mycelium;                                                    no change in plug.                                             Temperature require-                                                                         25° C.-Abundant growth; fair                            ments (ISP medium                                                                            aerial mycelium; reverse                                       No. 2 yeast extract                                                                          light brown; no soluble                                        malt extract slants)                                                                         pigment.                                                                      30° C.-Abundant growth; fair                                           aerial mycelium; reverse                                                      light brown; no soluble                                                       pigment.                                                                      37° C.-Abundant growth; fair                                           aerial mycelium; reverse-brown;                                               soluble pigment brown.                                                        40° C.-Abundant growth; sparse                                         aerial mycelium; reverse red                                                  brown; soluble pigment deep                                                   red brown.                                                                    45° C.-Fair growth; no aerial                                          mycelium; reverse red brown;                                                  moderate red brown pigment.                                    ______________________________________                                    

The results of carbon utilization tests carried out with organism NRRL8092 are set forth below.

    ______________________________________                                        Carbon Source   Response                                                      ______________________________________                                        D-glucose       +                                                             L-arabinose     +                                                             D-xylose        +                                                             D-fructose      +                                                             sucrose         -                                                             D-mannitol      -                                                             i-inositol      +                                                             rhamnose        +                                                             raffinose       -                                                             C control                                                                     (no carbohydrate)                                                                             -                                                             ______________________________________                                    

Certain characteristics of the A-28086-producing S. aureofaciens strainsdiffer from the characteristics of the organism described by Shirlingand Gottlieb. These differences are summarized in Table III:

                  TABLE III                                                       ______________________________________                                        Carbon                            Published                                   utilization                                                                             NRRL 5758   NRRL 8092   Description                                 ______________________________________                                        sucrose   -           -           +                                           i-inositol                                                                              +           +           -                                           rhamnose  +           +           -                                           Gelatin   30% in 14   50% in 14   Limited or                                  Liquefaction                                                                            days        days        None                                        Action on Milk                                                                          Milk pep-   Milk pep-   Limited and                                           tonized,    tonized,    variable                                              white       pale-yel-   peptoniza-                                            growth      low         tion (often                                           ring        growth      none);                                                            ring        limited                                                                       growth and                                                                    coagulation                                 ______________________________________                                    

The characteristics of organism NRRL 5758 which differ from thecharacteristics of organism NRRL 8092 are summarized in Table IV.

                  TABLE IV                                                        ______________________________________                                        Characteristic                                                                              NRRL 5758    NRRL 8092                                          ______________________________________                                        Vegatative Color                                                                            Yellow on    Cream to pale-                                                   several      yellow on                                                        media        several media                                      Sporulaton    Some spiral  Short straight                                                   sporophores  sporophores                                                      on tomato-   with occasional                                                  paste:oatmeal                                                                              hooks                                                            and inorganic                                                                 salts-starch                                                                  media                                                           Growth on:                                                                    Calcium malate                                                                              Growth fair, Growth sparse,                                                   brown; with  clear, no                                                        clearing     clearing                                           Inorganic salts-                                                                            Moderate spor-                                                                             Scant sporula-                                     starch        ulation;     tion aerial                                                      aerial pur-  pale yellow                                                      plish white  gray                                                             to gray.                                                        Bennett's agar                                                                              Reverse pale-                                                                              Reverse pink                                                     yellow                                                          ______________________________________                                    

The S. aureofaciens cultures useful for production of A-28086antibiotics have been deposited and made a part of the stock culturecollection of the Northern Marketing and Nurition Research Division,U.S. Dept. of Agriculture, Agricultural Research Service, Peoria, Ill.,61604, from which they are available to the public under the numbersNRRL 5758 and NRRL 8092.

The culture medium employed to grow S. aureofaciens NRRL 5758 or S.aureofaciens NRRL 8092 can be any one of a number of media. For economyin production, optimal yield, and ease of product isolation, however,certain culture media are preferred. Thus, for example, preferredcarbohydrate sources in large-scale fermentation are tapioca dextrin andsucrose, although glucose, corn starch, fructose, mannose, maltose,lactose, and the like can also be employed. Corn oil, peanut oil,soybean oil and fish oil are other useful sources of carbon. A preferrednitrogen source is enzyme-hydrolyzed casein, although peptones, soybeanmeal, cotton-seed meal, amino acids such as glutamic acid, and the likeare also useful. Among the nutrient inorganic salts which can beincorporated in the culture media are the customary soluble saltscapable of yielding sodium, magnesium, calcium, ammonium, chloride,carbonate, sulfate, nitrate, and like ions.

Essential trace elements necessary for the growth and development of theorganism should also be included in the culture medium. Such traceelements commonly occur as impurities in other constituents of themedium in amounts sufficient to meet the growth requirements of theorganism.

It may be necessary to add small amounts (i.e. 0.2 ml/l.) of an antifoamagent such as polypropylene glycol to large-scale fermentation media iffoaming becomes a problem.

For production of substantial quantities of the A-28086 antibiotics,submerged aerobic fermentation in tanks is preferred. Small quantitiesof the A-28086 antibiotics may be obtained by shake-flask culture.Because of the time lag in antibiotic production commonly associatedwith inoculation of large tanks with the spore form of the organism, itis preferable to use a vegetative inoculum. The vegetative inoculum isprepared by inoculating a small volume of culture medium with the sporeform or mycelial fragments of the organism to obtain a fresh, activelygrowing culture of the organism. The vegetative inoculum is thentransferred to a larger tank. The medium used for the growth of thevegetative inoculum can be the same as that employed for largerfermentations, but other media can also be employed.

The A-28086-producing organisms can be grown at temperatures betweenabout 20° and about 40° C. Optimum A-28086 production appears to occurat temperatures of about 27°-30° C.

As is customary in aerobic submerged culture processes, sterile air isblown through the culture medium. For efficient growth of the organismthe volume of air employed in the tank production is preferably above0.1 volume of air per volume of culture medium per minute. For efficientproduction of the A-28086 antibiotics the volume of air employed in thetank production is preferably above 0.25 volume of air per volume ofculture medium per minute. High levels of dissolved oxygen do notdepress antibiotic production.

The initial pH of the uninoculated culture medium varies with the mediumused. In general, the pH should be in the range of 6.0 to 7.5. Theharvest pH at the end of the fermentation is usually slightly higher, inthe range of 6.5 to 8.0.

Generally, antibiotic activity is detectable on the second day of thefermentation. Maximum production of antibiotic activity usually occursbetween about the sixth and the tenth days.

Following their production under submerged aerobic fermentationconditions, the A-28086 antibiotics previously described can berecovered from the fermentation medium by methods commonly employed inthe fermentation art. The antibiotic activity produced duringfermentation of an A28086-producing organism occurs in both the mycelialmass and in the filtered broth. Maximum recovery of the A-28086antibiotics is accomplished, therefore, by a combination of methods,including filtration, extraction, and adsorption chromatography. Apreferred solvent for separating the A-28086 antibiotics from eitherwhole or filtered fermentation broth is ethyl acetate, although othercommonly used solvents are satisfactory.

An especially advantageous method of separating the A-28086 factors A, Band D is to lower the pH of the whole fermentation broth to about pH3.0. At this pH the A-28086 factors A, B, and D are convenientlyseparated with the mycelial mass by filtration. Another advantageousmethod of separating the A-28086 factors involves adding a bicarbonatesuch as, for example, sodium bicarbonate, to the whole broth in amountsof approximately one gram per liter. The A-28086 factors are, thereby,conveniently separated with the mycelial mass in salt form. Methanol isa preferred solvent for separating the antibiotics from the mycelialmass, but other lower alcohols and ketones are also suitable.

Azeotropic distillation can also be advantageously employed in therecovery of the A-28086 antibiotics. In this method an organic solventwhich forms an appropriate azeotrope with water is added to the aqueousfermentation broth. This solvent-broth mixture is subjected toazeotropic distillation in order to remove at least half the water fromthe broth, leaving a water-solvent mixture in which the A-28086antibiotics are in solution in the organic solvent. Insolubleby-products can be separated by suitable means such as filtration orcentrifugation. The A-28086 antibiotics can then be recovered from theorganic solution by well-known procedures such as evaporation ofsolvent, precipitation by adding a nonsolvent, or extraction.

Further purification of the A-28086 antibiotics includes additionalextraction and adsorption procedures. Adsorptive materials such assilica gel, carbon, Florisil® (magnesium silicate, Floridin Co., P.O.Box 989, Tallahassee, Fla.) and the like can be advantageously employed.

Alternatively, the culture solids, including medium constituents andmycelium can be used without extraction or separation, but preferablyafter removal of water, as a source of the A-28086 antibiotics. Forexample, after production of A-28086 antibiotic activity, the culturemedium can be dried by lyophilization and mixed directly into feedpremix.

In another aspect, after production of A-28086 activity in the culturemedium, the mycelium can be separated and dried to give a product whichcan be used directly in a feed premix.

Under the conditions employed thus far, the S. aureofaciens strainsdescribed previously and designated as NRRL 5758 and NRRL 8092 produceantibiotic A-28086 factor A as the predominant factor. Although theratio of factors varies depending on the fermentation conditions used,in general factor A accounts for more than 99 percent of the totalrecovered antibiotic activity from NRRL 5758 and for about 90 percent ofthe total recovered antibiotic activity from NRRL 8092. A-28086 factor Baccounts for most of the remianing antibiotic activity from NRRL 5758,and factor D is a minor factor. On the other hand, A-28086 factor Daccounts for about 8-10 percent of the total recovered antibioticactivity from NRRL 8092, and factor B is a minor factor.

Antibiotic A-28086 factors A, B, and D are separated from each other andare isolated as individual compounds by the use of well-known methodssuch as column chromotography, thin-layer chromatography and the like.For example, column chromatography over silica gel is used to separatefactors A, B, and D by eluting the column with varying solvent mixtures,such as benzene-ethyl acetate. Using benzene-ethyl acetate solventmixtures over a silica gel column, factor B is eluted first, and factorsA and D are eluted later. Thin-layer chromatography, as describedhereinabove, is a convenient method for monitoring elution progress.

EXAMPLE 1 A. Shake-flask Fermentation of A-28086 using S. aureofaciensNRRL 5758

A culture of S. aureofaciens NRRL 5758 was prepared and maintained in anagar slant having the following composition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Agar                 20      g                                                Dextrin              10      g                                                Enzyme-hydrolyzed                                                             casein               2       g                                                Beef extract         2       g                                                Yeast extract        2       g                                                Distilled water      q.s. 1  liter                                            ______________________________________                                    

The slant was inoculated with S. aureofaciens NRRL 5758, and theinoculated slant was incubated at 30° C. for 6 to 10 days. The matureslant culture was covered with beef serum, and scraped with a sterileloop to loosen the spores. The resulting beef-serum suspension of sporesand mycelial fragments was lyophilized into six pellets.

One lyophilized pellet thus prepared was used to inoculate 50 ml of avegetative medium having the following composition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Glucose              20      g                                                Soybean grits        15      g                                                Corn-steep liquor    10      g                                                CaCO.sub.3           2       g                                                Tap Water            q.s. 1  liter                                            ______________________________________                                    

The inoculated vegetative medium, in a 250-ml Erlenmeyer flask, wasincubated at 30° C. for 72 hours on a shaker rotating through an arc 2inches in diameter at 250 rpm.

B. Tank Fermentation of A-28086 using S. aureofaciens NRRL 5758

In order to provide a larger volume of inoculum, 10 ml of the incubatedvegetative medium described above was used to inoculate 400 ml of asecond-stage vegetative growth medium having the same composition asthat of the vegetative medium. This second-stage medium, in a 2-literflask, was incubated at 30° C. for 24 hours on a shaker rotating throughan arc 2 inches in diameter at 250 rpm.

This second-stage vegetative medium (1 liter) was used to inoculated 100liters of sterile production medium of the following composition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Tapioca dextrin      60.0    g/l                                              Enzyme-hydrolyzed                                                             casein               8.0     g/l                                              Molasses             15.0    g/l                                              MgSO.sub.4 . 7H.sub.2 O                                                                            0.5     g/l                                              CaCO.sub.3           2.0     g/l                                              Refined soybean oil  5.0     g/l                                              Deionized water      q.s. 1  liter                                            ______________________________________                                    

The pH of the medium was 6.7 after sterilization by autoclaving at 120°C. for 30 minutes at 15-20 pounds pressure. In a 165-liter fermentationtank, the inoculated production medium was allowed to ferment for 10days at a temperature of 29° C. The fermentation medium was aerated withsterile air at the rate of 0.4 volume of air per volume of culturemedium per minute. The medium was stirred with conventional agitators at250 rpm.

EXAMPLE 2

The A-28086 antibiotics were produced according to the process ofExample 1, but utilizing a flask-production medium having the followingcomposition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Glucose              10      g/l                                              Edible molasses      20      g/l                                              Peptone              5       g/l                                              CaCO.sub.3           2       g/l                                              Tap Water            q.s. 1  liter                                            ______________________________________                                    

Separation of the A-28086 Antibiotic Complex Produced by S. aureofaciensNRRL 5758

Whole fermentation broth (132 liters), obtained by the method describedin Example 1, as filtered with a filter aid (Hyflo Super-cel, adiatomaceous earth, Johns-Manville Products Corp.) to give 97 liters offiltered broth. The filered broth was extracted with an approximatelyequal volume of ethyl acetate. The ethyl acetate extract was separatedfrom the aqueous phase and was concentrated to a volume of about 500 ml.This concentrated ethyl acetate extract was added to a large excess ofpetroleum ether (Skellysolve F; about 10 liters) to precipitate and,thereby, separate unwanted material. The separated filtrate wasevaporated under vacuum to give the broth portion of A-28086 antibioticcomplex (6.9 g).

The mycelial portion of A-28086 antibiotic complex was obtained byextracting the filtered mycelium twice with approximately half volumesof methanol (62 liters and 59 liters). The two methanol extracts werecombined and were concentrated under vacuum to remove the methanol.After this concentration about 10 liters of an aqueous phase remained.This aqueous phase was adjusted to about pH 7.5 with dilute sodiumhydroxide. The resulting solution was extracted twice with approximatelyequal volumes of ethyl acetate (9 liters and 10 liters). The ethylacetate extracts were combined and then concentrated to a volume ofabout 400 ml. This concentrated ethyl acetate extract was added to alarge excess of petroleum ether to remove unwanted materials, using theprocedure described above for the concentrated filtered broth extract.The mycelial portion of A-28086 antibiotic complex obtained from thefiltrate weighed 20.6 g.

EXAMPLE 4 Isolation of A-28086 Individual Factors A and B

The mycelial portion of A-28086 antibiotic complex (235 g, prepared asdescribed in Example 3) was dissolved in about 80 ml of benzene. Thisbenzene solution was applied to a silica gel column (9 × 130 cm, 8liters, Matheson grade 62 silica gel). The column was eluted withvarying benzene-ethyl acetate mixtures. Elution progress was followed bythin-layer chromatography. Using a benzene-ethyl acetate (90:10) solventsystem, factor B was eluted first and was isolated as an individualfactor. Factor B (43 mg) was crystallized from acetone-water, m.p.150°-153° C.

Continuing to elute with benzene-ethyl acetate mixtures but graduallyincreasing the ratio of ethyl acetate present, factor A was eluted; thevarious fractions containing factor A were combined and wereconcentrated under vacuum to a residue. This residue was dissolved inacetone (about 150 ml); water (about 150 ml) was added to the acetonesolution. The pH of the resulting solution was adjusted to pH 3 by theaddition of 1N hydrochloric acid. The acidified mixture was stirredabout one hour, during which time a precipitate formed. This precipitatewas separated by filtration and was recrystallized from acetone (about150 ml) upon addition of water (about 60 ml). The product was driedovernight under vacuum to give factor A (about 6.6 g). After partialevaporation of acetone from the filtrate, a second crop of factor A(about 1.2 g) was obtained.

EXAMPLE 5 A-28086 Factor A-Acetyl Ester Derivative

Antibiotic A-28086 factor A (7.4 g) was dissolved in pyridine (150 ml).Acetic anhydride (50 ml) was added to this solution. The resultingsolution was mixed thoroughly and then was allowed to stand overnight atroom temperature.

Water (200 ml) was added, mixing thoroughly. This mixture was allowed tostand for 4 hours at room temperature. A white solid precipitated; thissolid was separated by filtration, washed with water, and air dried. Theresulting solid was dissolved in acetone (100 ml); and the acetonesolution was evaporated to dryness under vacuum (this was repeated threetimes). The residue thus obtained crystallized from acetone (100ml)-water (50 ml), to give A-28086 factor A acetyl derivative (6.14 g),melting point 100°-103° C.

EXAMPLES 6-9

Antibiotic A-28086 factor A propionyl ester derivative, prepared byreacting factor A with propionic anhydride in the presence of pyridineaccording to the method of Example 5, melting point 96°-98° C.

Antibiotic A-28086 factor A n-butyryl ester derivative, prepared byreacting factor A with n-butyric anhydride in the presence of pyridineaccording to the method of Example 5, melting point 79°-81° C.

Antibiotic A-28086 factor A n-caproyl ester derivative, prepared byreacting factor A with caproic anhydride in the presence of pyridineaccording to the method of Example 5, melting point 163°-167° C.

Antibiotic A-28086 factor A n-valeryl ester derivative, prepared byreacting factor A with valeric anhydride in the presence of pyridineaccording to the method of Example 5, melting point 173°-175° C.

EXAMPLE 10 Preparation of A-28086 Factor A Sodium Salt

Antibiotic A-28086 factor A (500 mg) was dissolved in acetone (50 ml).Water (50 ml) was added to this solution, and 5N sodium hydroxide wasadded to bring the pH of the solution to 10.5-11. The resulting solutionwas stirred for 1 hour and then extracted with ethyl acetate. The ethylacetate extract was evaporated to dryness under vacuum. The residue wasprecipitated from an acetone-water solution to give 378 mg of A-28086factor A sodium salt, melting point 120°-123° C.

EXAMPLES 11-15

Antibiotic A-28086 factor A barium salt was prepared from antibioticA-28086 factor A (500 mg) and saturated barium hydroxide, using themethod of Example 10 to give 369 mg of A-28086 factor A barium salt,melting point 188°-190° C.

Antibiotic A-28086 factor A potassium salt was prepared from antibioticA-28086 factor A (500 mg) and 5N potassium hydroxide, using the methodof Example 10 to give 363 mg of A-28086 factor A potassium salt, meltingpoint 165°-167° C.

Antibiotic A-28086 factor A cesium salt was prepared from antibioticA-28086 factor A (500 mg) and 1N cesium hydroxide, using the method ofExample 10 to give 540 mg of A-28086 factor A cesium salt, melting point190°-210° C.

Antibiotic A-28086 factor B sodium salt, prepared from antibioticA-28086 factor B and 5N sodium hydroxide according to the method ofExample 10.

EXAMPLE 16 Shake-flask Fermentation of A-28086 using S. aureofaciensNRRL 8092

A culture of S. aureofaciens NRRL 8092 was prepared and maintained on anagar slant having the following composition:

    ______________________________________                                        Ingredient           Amount                                                   ______________________________________                                        K.sub.2 HPO.sub.4    2 g                                                      MgSO.sub.4 . 7H.sub.2 O                                                                            0.25 g                                                   NH.sub.4 NO.sub.3    2 g                                                      CaCO.sub.3           2.5 g                                                    FeSO.sub.4 . 7H.sub.2 O                                                                            0.001 g                                                  MnCl.sub.2 . 7H.sub.2 O                                                                            0.001 g                                                  ZnSO.sub.4 . 7H.sub.2 O                                                                            0.001 g                                                  Glucose              10 g                                                     Agar                 20 g                                                     Deionized water-     q.s. 1 liter                                             pH (unadjusted)      7.7                                                      ______________________________________                                    

The slant was inoculated with S. aureofaciens NRRL 8092, and theinoculated slant was incubated at 30° C for about 7 days. The matureslant culture was covered with sterile beef serum and was scraped with asterile loop to prepare a spore and mycelial suspension from the slantculture. The resulting suspension was lyophilized into a maximum of sixpellets.

One of the lyophile pellets thus prepared was used to inoculate 50 ml ofa vegetative medium having the following composition:

    ______________________________________                                        Ingredient           Amount                                                   ______________________________________                                        Glucose              20 g                                                     Soybean flour        15 g                                                     Corn-steep liquor    10 g                                                     CaCO.sub.3            2 g                                                     Tap Water            q.s. 1 liter                                             pH adjusted to pH 6.5 with dil NaOH                                           ______________________________________                                    

The inoculated vegetative medium, in a 250-ml Erlenmeyer flask, wasincubated at 30° C. for 48 hours on a rotary shaker at 250 rpm with a2-inch arc.

The incubated vegetative medium described above (0.5 ml, 1 percent) wasused to inoculate 50 ml of a fermentation medium having the followingcomposition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Tapioca dextrin*      60.0    g                                               Enzyme-hydrolyzed casein**                                                                          6.0     g                                               Enzymatic hydrolysate of                                                      casein***             2.0     g                                               CaCO.sub.3            2.0     g                                               MgSO.sub.4 . 7H.sub.2 O                                                                             0.5     g                                               Blackstrap molasses   15.0    g                                               Refined soybean oil   5.0     ml                                              Tap Water             q.s. 1 liter                                            pH (unadjusted) 6.6                                                           ______________________________________                                          *Staley Dextrin No. 11, A. E. Staley Co., Decatur, Ill.                       **Amber EHC, Amber Laboratories, Juneau, Wisc.                                ***NZ Amine A, Sheffield Chemical Co., Norwich, N.Y.                    

EXAMPLE 17 Tank Fermentation of A-28086 using S. aureofaciens NRRL 8092

The initial procedure described in Example 16 for the shake-flaskfermentation of A-28086 was also used for tank fermentation. In order toproduce a larger volume of inoculum, 10 ml of the incubated vegetativemedium was used to inoculate 400 ml of a second-stage vegetative mediumhaving the same composition as that of the first vegetative medium. Thissecond-stage medium, in a 2-liter Erlenmeyer flask, was incubated at 30°C. for 24 hours on a rotary shaker at 250 rpm with a 2-inch arc.

This incubated second-stage vegetative medium (800 ml) was used toinoculate 100 liters of sterile fermentation medium having the followingcomposition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Tapioca dextrin*      60.0    g/l                                             Enzyme-hydrolyzed casein**                                                                          6.0     g/l                                             Enzymatic-hydrolysate of                                                      casein***             2.0     g/l                                             CaCO.sub.3            2.0     g/l                                             MgSO.sub.4 . 7H.sub.2 O                                                                             0.5     g/l                                             Blackstrap molasses   15.0    g/l                                             Refined soybean oil   5.0     mg/l                                            Tap Water             q.s. 1 liter                                            ______________________________________                                          *Staley Dextrin No. 11, A. E. Staley Co., Decatur, Ill.                       **Amber EHC, Amber Laboratories, Juneau, Wisc.                                ***NZ Amine A, Sheffield Chemical Co., Norwich, N.Y.                    

The pH of the medium was 6.8 ± 0.1 after sterilization by autoclaving at121° C. for 30 minutes at 15-20 pounds pressure. In a 165-literfermentation tank, the inoculated production medium was allowed toferment for 10-12 days at 28 ± 1° C. The fermentation medium was aeratedwith sterile air at the rate of 0.4 volume of air per volume of culturemedium per minute. The medium was stirred with conventional agitators at300 rpm.

EXAMPLE 18 Separation of the A-28086 Antibiotic Complex Produced by S.aureofaciens NRRL 8092

Whole fermentation broth (60 liters), obtained by the method describedin Example 17, was adjusted to pH 3 by the addition of dilute HCl. Theresulting solution was filtered using a filter aid (Hyflo Super-cel, adiatomaceous earth, Johns-Manville Products Corp.). The separatedmycelial cake was extracted with 30 liters of methanol, adding 1.56 kgof NaHCO₃ to the extract with stirring. After separation of thisextract, the mycelial cake was again extracted with another 30 liters ofmethanol. The two methanol extracts were combined and concentrated undervacuum to remove the methanol. The remaining aqueous solution (about 7liters) was adjusted to pH 7.5 with dilute HCl. The resulting solutionwas extracted twice with ethyl acetate (7-liter portions). The ethylacetate extracts were combined and concentrated under vacuum to given anoily residue. This oily residue was dissolved in 1500 ml of acetone.Water (1500 ml) was added to the acetone solution. The resultingsolution was adjusted to pH 3 with dilute HCl and was stirred one hour.The precipitate which had formed was separated by filtration and thenwas dissolved in acetone (1500 ml); water (400 ml) was added to thissolution. The resulting solution was allowed to stand for 16 hours forcrystallization to occur. The crystals formed were separated byfiltration and dried under vacuum to give 74 g crude crystalline productcontaining A-28086 factors A, B and D and crystalline impurities.

This crude crystalline product (40 g) was dissolved in about 250 ml ofbenzene. The benzene solution was then applied to a silica-gel column(9- × 120-cm column; Grace-Davidson grade 62 silica gel). The column waseluted successively with 40 liters of each of the following:

(1) benzene

(2) benzene:ethyl acetate (9:1)

(3) benzene:ethyl acetate (4:1)

(4) benzene:ethyl acetate (7:3)

(5) benzene:ethyl acetate (1:1)

(6) ethyl acetate

(7) methanol

One-liter fractions were collected. Each fraction was checked by assayagainst Bacillus subtilis and by thin-layer chromatography to identifythe eluted compounds. A-28086I was eluted with benzene:ethyl acetate(4:1). A-28086 factor B was eluted with benzene:ethyl acetate (7:3).A-28086 factors A and D were eluted in the fractions obtained withbenzene:ethyl acetate (7:3 and 1:1), fractions 119-156. These fractionswere combined and evaporated to dryness under vacuum. The residue thusobtained was dissolved in acetone (500 ml). Water (500 ml) was added tothe acetone solution, and the resulting solution was adjusted to pH 3with dilute HCl and was stirred for one hour. The precipitate whichformed was separated by filtration and was crystallized from acetone(500 ml)-water (180 ml). The crystals thus formed were separated byfiltration and dried under vacuum to give 20.1 g of a mixture of A-28086factors A and D.

EXAMPLE 19 Separation and Purification of Individual Factors A and D

The crystalline mixture of A-28086 factors A and D obtained in Example18 (18.8 g) was dissolved in benzene (50 ml). The benzene solution wasapplied to a silica-gel column (7- × 100-cm column; E. Merck grade 60silica gel, finer than 230 mesh ASTM). The column was eluted, at a flowrate of 90 ml per hour, successively with:

(1) 12 liters of benzene

(2) 12 liters of benzene:ethyl acetate (9:1)

(3) 12 liters of benzene:ethyl acetate (4:1)

(4) 32 liters of benzene:ethyl acetate (7:3)

(5) 10 liters of methanol

One-to two-liter fractions were collected until activity was detected;then 200-ml fractions were collected. The fractions containing onlyA-28086 factor D were combined and evaporated under vacuum to a residue.This residue crystallized from acetone-water (1:1). The crystals wereseparated and dried under vacuum to give 140 mg of crystalline A-28086factor D.

The fractions containing A-28086 factor D with a trace of A-28086 factorA were treated in the same manner to give an additional 150 mg ofcrystalline A-28086 factor D containing a small amount of A-28086 factorA.

The fractions containing only A-28086 factor A were also treated in thesame manner to give 4.7 g of crystalline A-28086 factor A.

EXAMPLE 20 Preparation of A-28086 Factor D Sodium Salt

Antibiotic A-28086 factor D is dissolved in acetone. An equivalentamount of water is added to this solution, and sufficient 5N sodiumhydroxide is added to bring the pH of the solution to about pH 11. Theresulting solution is stirred for about an hour and then is extractedwith ethyl acetate. The ethyl acetate extract is evaporated under vacuumto give A-28086 factor D sodium salt.

EXAMPLES 21-23

Antibiotic A-28086 factor D potassium salt, prepared from A-28086 factorD and 5N potassium hydroxide, using the method of Example 10.

Antibiotic A-28086 factor D barium salt, prepared from A-28086 factor Dand saturated barium hydroxide, using the method of Example 10.

Antibiotic A-28086 factor D cesium salt, prepared from A-28086 factor Dand 1N cesium hydroxide, using the method of Example 10.

Further ionophorous coccidiocides exemplary of those which may be usedin the practice of this invention include Al50, U.S. Pat. No. 3,711,605;salinomycin, U.S. Pat. No. 3,857,948; dianemycin, U.S. Pat. No.3,577,531; and X206, U.S. Pat. No. 3,794,732.

The ionophorous coccidiocides used in this invention have hydroxylgroups, which may be esterified to form acyl esters. Such esters,particularly the C₂ -C₆ alkanoyl esters, are equivalent to thecoccidiocides and may be used in the practice of the present invention.Esterification occurs at one or more of the hydroxyl groups upon simpletreatment with a C₂ -C₆ carboxylic acid anhydride or acid halide, forexample, at room temperature in methanol.

The compounds used in this invention, and the acyl esters thereof, formphysiologically-acceptable salts. Accordingly, thephysiologically-acceptable alkali metal, alkaline earth metal and aminesalts of the compounds and of their C₂ -C₆ alkanoyl ester derivativesare also used in this invention. "Physiologically-acceptable" salts aresalts in which the toxicity of the compound as a whole towardwarm-blooded animals is not increased relative to the non-salt form.Representative alkali-metal and alkaline-earth-metal salts include thesodium, potassium, lithium, cesium, rubidium, barium, calcium andmagnesium salts.

Suitable amine salts include the ammonium and the primary, secondary andtertiary C₁ -C₄ alkylamine and C₂ -C₄ hydroxyalkylamine salts.Illustrative amine salts include those formed by reaction of thecoccidiocides with ammonium hydroxide, methylamine, s-butylamine,isopropylamine, diethylamine, diisopropylamine, ethanolamine,triethylamine, 3-amino-1-propanol and the like.

The salts of the coccidiocides are prepared according to the generalprocedures commonly employed for the preparation of cationic salts. Forexample, the free acid form of the compound is dissolved in a suitablesolvent, and an aqueous or organic solvent solution of the desired baseis added to the coccidiocide solution. The salts are isolated byfiltration or by evaporation of the solvent.

It is well known in the veterinary pharmaceutical art that conditionswithin the treated animal or bird frequently change a compound tochemical forms other than that in which it was administered. Therefore,the form in which it may be administered does not affect the method oftreatment and may be chosen for reasons of economics or convenience. Allof the compounds described herein, therefore, may be administered in theform of any desired alkanoyl ester or salt without effect on theefficacy of the invention.

The following exemplary coccidiocides are typical of the alkanoyl esterand salt forms in which the compounds of this invention may be used. Thecompounds are mentioned only to assure that the reader fully understandsthe invention, and are not to be interpreted as bounding the scope ofthe usable compounds.

monensin diacetate

nigericin propionate, lithium salt

A204 acetate, magnesium salt

A28086, sodium salt

A28695 butyrate

lasalocid acetate, ammonium salt

A204 pentanoate

monensin, potassium salt

A204 acetate, ethylamine salt

nigericin, ethanolamine salt

A28086 hexanoate

A28695, triethylamine salt

monensin propionate, s-butylamine salt

A28695 acetate

monensin, diethylamine salt

A204 pentanoate, potassium salt

nigericin, calcium salt

A28695, barium salt

lasalocid propionate, methylamine salt

lasalocid, ammonium salt

monensin, isopropanolamine salt

nigericin, dimethylamine salt

A28695, magnesium salt

A28695 hexanoate

A28086 propionate

A28086, calcium salt

The present invention has been tested by in vivo experiments withchickens infected with coccidiosis. The following tests are exemplary ofthe efficacy of various embodiments of the invention.

In the tests described below, the coccidiocides were used as theunresolved mixtures of factors produced by fermentation. All of thecompounds, except A28086, were in the form of sodium or mixedsodium-potassium salts. A28086 was used as the free acid.

Untreated, infected control birds and untreated normal control birdswere used in all experiments described below. The birds used were from ahomogeneous research flock, and the E. tenella culture used to infectthe birds was from a laboratory strain known to reproduce consistentlyand to produce uniform infections. Each bird was orally inoculated with20,000-40,000 sporulated oocysts. The coccidiocides were mixed with thebirds' feed in concentrations, measured in ppm., shown in the tablesbelow.

The extent of coccidial infection is expressed as a lesion score on anarbitrary 0-4 scale. At the end of the tests, all birds were killed, andtheir intestinal tracts were examined. Birds which showed none of thelesions left by coccidia were scored 0, and birds with extremely severeinfections were scored 4. Intermediate degrees of infection were givenlesion scores of 1, 2 or 3. The scores of all the birds which received agiven treatment were averaged.

TEST 1

monensin + A204

One-week-old broiler cockerels were allotted to 10-bird cages and werefed medicated or control ration for one day prior to infection with E.tenella. The birds were maintained on the same rations for seven daysand infections were scored as described above. Each cage constituted atreatment group.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.9                                                monensin      50           2.1                                                A204           5.5         3.3                                                A204          16.5         0.6                                                monensin                                                                      + A204        50 + 5.5     1.4                                                monensin                                                                      + A204        50 + 16.5    0                                                  ______________________________________                                    

TEST 2

monensin + A204

This test was conducted in the same fashion as the test above, eaceptthat each treatment group comprised five birds.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.0                                                monensin      50           2.4                                                A204           5.5         3.0                                                A204          7.7          2.6                                                A204          12.1         1.6                                                A204          16.5         0.2                                                monensin +                                                                    A204          50 + 5.5     0.4                                                monensin +                                                                    A204          50 + 7.7     0.4                                                monensin +                                                                    A204          50 + 12.1    0.4                                                monensin +                                                                    A204          50 + 16.5    0                                                  ______________________________________                                    

TEST 3

monensin + A204

The procedure in this test followed the procedure of Test 1.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.3                                                monensin      25           3.4                                                monensin      50           2.5                                                A204           5           3.1                                                A204          10           1.6                                                monensin +                                                                    A204          25 + 5       2.7                                                monensin +                                                                    A204          25 + 10      1.2                                                monensin +                                                                    A204          50 + 5       1.9                                                monensin +                                                                    A204          50 + 10      0.9                                                ______________________________________                                    

TEST 4 monensin + A204

This test followed the procedure of Test 1, except that each treatmentgroup consisted of three cages of 10 birds each.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           2.9                                                monensin      50           1.7                                                A204           5.5         2.3                                                monensin +                                                                    A204          50 + 5.5     0.6                                                ______________________________________                                    

TEST 5

monensin + A204

The procedure of this test followed that of Test 1 again, except thateach treatment group comprised 5 replicates of 5 birds per cage.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.0                                                monensin      50           2.2                                                A204           7           2.2                                                monensin +                                                                    A204          50 + 7       0.9                                                ______________________________________                                    

TEST 6

monensin + nigericin

In this test, the procedure of Test 1 was used.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.4                                                monensin       50          2.2                                                nigericin     100          2.8                                                monensin +                                                                    nigericin      50 + 100    0.4                                                ______________________________________                                    

TEST 7

monensin + nigericin

The procedure of Test 5 was used.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.0                                                monensin       50          2.2                                                nigericin     100          2.4                                                monensin +                                                                    nigericin      50 + 100    0.5                                                ______________________________________                                    

TEST 8

monensin + A28086

The procedure of Test 1 was used.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.4                                                monensin      50           2.2                                                A28086        42           2.8                                                monensin +                                                                    A28086        50 + 42      1.4                                                ______________________________________                                    

TEST 9

monensin + A28086

The procedure of Test 1 was used.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.3                                                monensin      25           3.4                                                monensin      50           2.5                                                A28086        40           2.4                                                monensin +                                                                    A28086        25 + 40      1.3                                                monensin +                                                                    A28086        50 + 40      1.0                                                ______________________________________                                    

TEST 10

monensin + A28086

The procedure of Test 5 was used.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.0                                                monensin      50           2.2                                                A28086        45           2.5                                                monensin +                                                                    A28086        50 + 45      0.6                                                ______________________________________                                    

TEST 11

monensin + A28695

The procedure of Test 1 was used.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.4                                                monensin      50           2.2                                                A28695        15           3.2                                                monensin +                                                                    A28695        50 + 15      0.4                                                ______________________________________                                    

TEST 12

monensin + A28695

The procedure of Test 1 was used, except that each treatment groupconsisted of three cages of 10 birds each.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           2.7                                                monensin      50           1.7                                                A28695        15           2.5                                                monensin +                                                                    A28695        50 + 15      0.6                                                ______________________________________                                    

TEST 13

monensin + A28695

The procedure of Test 5 was used.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.0                                                monensin      50           2.2                                                A28695        15           2.6                                                monensin +                                                                    A28695        50 + 15      0.8                                                ______________________________________                                    

TEST 14

monensin + lasalocid

The procedure of Test 1 was followed, except that the chicks were orallyinfected with 60,000 E. tenella oocysts per bird. Each treatment groupcomprised five replicates of five birds each.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.5                                                monensin      50           3.2                                                lasalocid     37           3.3                                                monensin +                                                                    lasalocid     50 + 37      0.6                                                ______________________________________                                    

TEST 15

A28086 + a28695

the procedure of Test 1 was followed.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None             0         3.4                                                A28086          42         2.8                                                A28695          15         3.2                                                A28086 +                                                                      A28695          42 + 15    1.7                                                ______________________________________                                    

TEST 16

A204 + a28695

the procedure of Test 5 was followed.

    ______________________________________                                        Treatment     Concentration                                                                              Lesion Score                                       ______________________________________                                        None           0           3.0                                                A204           7           2.2                                                A28695        15           2.6                                                A204 +                                                                        A28695         7 + 15      0.7                                                ______________________________________                                    

The present invention is useful to protect poultry in general. Not onlysuch barnyard fowl as chickens, turkeys, ducks and geese may beprotected thereby, but also exotic fowl such as pheasants and quail.

Coccidiosis is likely to affect poultry of any age and in any type ofcultural practice. The present invention, therefore, may be used at anytime to protect poultry from coccidiosis. As noted above, however,coccidiosis is most likely to affect poultry being reared in confinedquarters, as in broiler houses and the like. Use of the invention inpoultry reared under such conditions, therefore, will provideparticularly great benefits.

Since coccidiosis is likely to break out at any time, use of thisinvention is recommended at any time of year and may be continued forany length of time. Continuous administration of a composition of thisinvention throughout the lives of the poultry is a particularly usefulembodiment of the invention. However, the administration of acomposition of this invention for even short periods of time, such as afew days, will also produce benefits and is a useful manner in which tocarry out the method of this invention.

The compositions and methods of this invention are used for the controlof coccidiosis in the usual fashion. Coccidiosis of poultry directlyaffects the intestinal tract, and anticoccidial drugs are thereforeadministered orally. The compositions of this invention, accordingly,are compositions wherein the synergistic coccidiocides are combined witheither a poultry feedstuff or with drinking water. The compositions arenovel only because of the presence therein of the synergisticcoccidiocides, and otherwise follow conventional practices in thefeedstuff industry.

The concentrations of the coccidiocides which are named herein are basedon food or drinking water compositions which are the entire food ordrinking water supply of the poultry. This method of describing theconcentrations of the coccidiocides is in accordance with the practiceof the poultry industry, since it is normal to supply to poultry onlyone source of food and one source of drinking water. Those of skill inpoultry husbandry will recognize, however, that the concentrations mustbe adjusted upward, should it be desirable to supply the poultry withvarious sources of food or water, only part of which are compositions ofthis invention.

Poultry feedstuffs made according to all formulae and manners used inthe poultry industry are perfectly satisfactory as carriers for thesynergistic coccidiocides of this invention. The following poultryfeedstuff formulae are representative of formulae typically used in theindustry and are listed to assist the reader. A poultry scientist orgrower will understand from inspection of the following formulae,however, that the formulation of the poultry feedstuff is not a limitingfactor in the use of this invention. Feedstuffs based on any grain andcontaining any vitamin concentrate, mineral concentrate or other drugsand feed additives are satisfactory. Both conventional dry and pelletedfeeds and liquid suspension feeds including feeds based on distiller'swastes and milk byproducts may be used in the compositions of thisinvention.

    ______________________________________                                        Turkey Starter                                                                Ingredients               Percent                                             ______________________________________                                        soybean meal, solvent extracted,                                              dehulled                  40.7                                                corn, yellow, ground      39.7                                                fish meal with solubles   5.0                                                 beef tallow               5.0                                                 corn distillers dried solubles                                                                          2.5                                                 alfalfa meal, dehydrated (17%)                                                                          2.5                                                 dicalcium phosphate feed grade                                                                          2.5                                                 calcium carbonate         1.2                                                 vitamin premix.sup.1      0.5                                                 salt (NaCl)               0.2                                                 trace mineral premix.sup.2                                                                              0.1                                                 methionine hydroxy analog 0.1                                                 Total                     100.0                                               Turkey Finisher                                                                Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      71.2                                                soybean meal, solvent extracted,                                              dehulled (50%)            9.9                                                 corn distillers dried solubles                                                                          5.0                                                 alfalfa meal, dehydrated (17%)                                                                          5.0                                                 animal fat                3.0                                                 fish meal with solubles   2.5                                                 dicalcium phosphate, feed grade                                                                         1.7                                                 calcium carbonate         0.5                                                 vitamin premix.sup.1      0.5                                                 salt (NaCl)               0.4                                                 methionine hydroxy analog 0.2                                                 trace mineral premix.sup.2                                                                              0.1                                                 Total                     100.0                                               Chick Starter, Light Breeds                                                    Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      56.3                                                soybean meal, solvent extracted,                                              dehulled (50%)            17.9                                                wheat middlings           10.0                                                corn distillers dried solubles                                                                          5.0                                                 fish meal with solubles   5.0                                                 alfalfa meal, dehydrated (17%)                                                                          2.5                                                 dicalcium phosphate, feed grade                                                                         1.3                                                 calcium carbonate         0.9                                                 vitamin premix.sup.1      0.5                                                 salt (NaCl)               0.3                                                 methionine hydroxy analog 0.2                                                 trace mineral premix.sup. 2                                                                             0.1                                                 Total                     100.0                                               Pullet Grower                                                                  Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      73.5                                                soybean meal, solvent extracted,                                              dehulled (50%)            21.9                                                dicalcium phosphate, feed grade                                                                         2.5                                                 calcium carbonate         1.0                                                 vitamin premix.sup.1      0.5                                                 salt (NaCl)               0.3                                                 methionine hydroxy analog 0.2                                                 trace mineral premix.sup.2                                                                              0.1                                                 Total                     100.0                                               Pullet Developer                                                               Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      67.5                                                oats, ground whole        15.0                                                soybean meal, solvent extracted,                                              dehulled (50%)            13.4                                                dicalcium phosphate, feed grade                                                                         2.1                                                 calcium carbonate         1.0                                                 vitamin premix.sup.1      0.5                                                 methionine hydroxy analog 0.3                                                 salt (NaCl)               0.2                                                 trace mineral premix.sup.2                                                                              0.1                                                 Total                     100.0                                               Layer, Light Breeds                                                            Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      61.00                                               calcium carbonate         8.50                                                soybean meal, solvent extracted,                                              dehulled (50%)            8.25                                                oats, ground whole        5.00                                                wheat middlings           5.00                                                corn distillers dried solubles                                                                          5.00                                                alfalfa meal, dehydrated (17%)                                                                          2.50                                                fish meal with solubles   2.00                                                dicalcium phosphate, feed grade                                                                         1.60                                                vitamin premix.sup.1      0.50                                                salt (NaCl)               0.30                                                methionine hydroxy analog 0.25                                                trace mineral premix.sup.2                                                                              0.10                                                Total                     100.00                                              Pullet Grower, Broiler Breeders                                                Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      38.1                                                oats, ground whole        30.0                                                soybean meal, solvent extracted,                                              dehulled (50%)            12.8                                                wheat middlings           10.0                                                alfalfa meal, dehydrated (17%)                                                                          5.0                                                 dicalcium phosphate, feed grade                                                                         1.7                                                 calcium carbonate         1.3                                                 vitamin premix.sup.1      0.5                                                 methionine hydroxy analog 0.3                                                 salt (NaCl)               0.2                                                 trace mineral premix.sup.2                                                                              0.1                                                 Total                     100.0                                               Pullet Developer, Broiler Breeders                                             Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      54.5                                                oats, ground whole        15.0                                                wheat middlings           10.0                                                alfalfa meal, dehydrated (17%)                                                                          7.5                                                 corn distillers dried solubles                                                                          5.0                                                 fish meal with solubles   2.5                                                 soybean meal, solvent extracted,                                              dehulled (50%)            1.9                                                 dicalcium phosphate, feed grade                                                                         1.4                                                 calcium carbonate         1.1                                                 vitamin premix.sup.1      0.5                                                 salt (NaCl)               0.3                                                 methionine hydroxy analog 0.2                                                 trace mineral premix.sup.2                                                                              0.1                                                 Total                     100.0                                               Broiler Breeder                                                                Ingredients              Percent                                             ______________________________________                                        corn, yellow, ground      42.8                                                oats, ground whole        25.0                                                soybean meal, solvent extracted,                                              dehulled (50%)            8.1                                                 wheat middlings           5.0                                                 corn distillers dried solubles                                                                          5.0                                                 fish meal with solubles   2.5                                                 alfalfa meal, dehydrated (17%)                                                                          2.5                                                 calcium carbonate         6.3                                                 dicalcium phosphate, feed grade                                                                         1.7                                                 vitamin premix.sup.1      0.5                                                 salt (NaCl)               0.3                                                 methionine hydroxy analog 0.2                                                 trace mineral premix.sup.2                                                                              0.1                                                 Total                     100.0                                               ______________________________________                                         .sup.1 vitamin premix provides 3000 IU of Vitamin A, 900 ICU of Vitamin D     40 mg of Vitamin E, 0.7 mg of Vitamin K, 1000 mg of choline, 70 mg of         niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 0.10 mg of Vitamin      B.sub.12, 0.10 mg of biotin and 125 mg of ethoxyquin per kg of complete       feed.                                                                         .sup.2 trace mineral premix provides 75 mg of manganese, 50 mg of zinc, 2     mg of iron and 1 mg of iodine per kg of complete feed.                   

It will be understood that poultry feed compositions according to thisinvention will usually be made by first preparing a concentrated premixwhich contains the synergistic coccidiocides in high concentrations,such as from about 0.25 percent to about 80 percent by weight. Suchpremixes comprise the coccidiocides dispersed in suchphysiologically-acceptable carriers as polyethylene glycols, propyleneglycol, inert oils including vegetable oils and highly-refined mineraloils, ethanol, water, aqueous alcohols, vermiculite, diatomaceous earth,attapulgite, cracked corn, soybean meal, alfalfa meal, rice hulls,ground corncob, and the like.

The synergistic coccidiocides of this invention are also readilyadministered in the drinking water of poultry. They are incorporatedinto drinking water by merely adding a water-soluble orwater-suspendible form of the compounds to water in the proper amount.Such compositions are most easily prepared by choosing a water-solubleform of the compounds. If an insoluble form is preferred, however, asuspension may be made. Suspensions are prepared by proper use ofphysiologically-acceptable adjuvants to keep the compounds in suspensionin the water. Adjuvants are chosen from among the thickeners, such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and the alginates.Many classes of surfactants also serve to suspend the compounds. Forexample, lecithin, alkylphenol polyethylene oxide adducts, naphthalenesulfonates, alkylbenzene sulfonates, and the polyoxyethylene sorbitanesters are useful for preparing suspensions. It is usually mostpractical to prepare a concentrated suspension, or a dry formulation ofthe synergistic coccidiocides and suspending agents, which concentratedformulation is diluted in plain drinking water for administration topoultry.

It will be understood by both chemists and poultry scientists that themethod of this invention may be combined with other methods of treatingand nourishing poultry. For example, a composition according to thepresent invention may be further fortified or medicated with growthpromoting agents, antibiotic drugs, parasiticides and the like withoutimpairing the efficacy of the present invention.

While the primary thrust of this invention is the protection of poultryfrom coccidiosis, it will be apparent that other animals may beprotected thereby, as well.

I claim:
 1. A coccidiocidal composition for poultry comprising acoccidiocidally-ineffective concentration of each of two ionophorousfermentation-derived coccidiocides, which coccidiocides aresynergistically coccidiocidally effective in combination, together witha poultry feedstuff or drinking water, wherein the coccidiocides and theconcentrations thereof are about 50 ppm of monensin and about 37 ppm oflasalocid.
 2. A coccidiocidal method for poultry which comprises orallyadministering to the poultry a coccidiocidally-effective combination ofabout 50 ppm of monensin and about 37 ppm of lasalocid, together with apoultry feedstuff or drinking water.